Cation binding properties of the multiple subforms of RVV-X, the coagulant protein from Vipera russelli.
The factor X activating enzyme from Russell's viper venom (RVV-X) has been shown to exist in multiple subforms, distinguished from each other by their isoelectric points. The differences in isoelectric points were due, as least in part, to dissimilarities in the respective sialic acid contents of the subforms. No functional difference was, however, discovered between any of the subforms. All of the subforms were found, by equilibrium ultrafiltration, to bind Ca2+ reversibly. At least two equivalent Ca2+ binding sites were observed on each protein molecule (Mr 79 000), with a KD of 50 +/- 15 microM at pH 7.4 and 25 degrees C. A new substrate for RVV-X, which does not bind Ca2+, apoprotein AI from human high-density lipoprotein, was used to show that this reversibly bound Ca2+ was not essential for enzymic activity. All subforms have also been shown, by atomic absorption analysis, to contain nonexchangeable metal ions, to the extent of 1 mol of Ca2+ and 0.7 mol of Zn2+ per mol of protein. No Mn2+ or Mg2+ was detected. This nonexchangeable Ca2+ and Zn2+ could only be removed from the protein by incubation at pH 3.0 or by treatment with 6 M guanidine hydrochloride, conditions under which the protein lost activity irreversibly.
D000818:Animals; D016632:Apolipoprotein A-I; D001053:Apolipoproteins / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D005170:Factor X / Q000378:metabolism; D006801:Humans; D007526:Isoelectric Point; D008666:Metalloendopeptidases; D011480:Protease Inhibitors; D014757:Viper Venoms / Q000032:analysis*; D015032:Zinc / Q000378:metabolism*
D016632:Apolipoprotein A-I; D001053:Apolipoproteins; D002413:Cations, Divalent; D002614:Chelating Agents; D011480:Protease Inhibitors; D014757:Viper Venoms; D005170:Factor X; D010450:Endopeptidases; D008666:Metalloendopeptidases; C033858:russellysin; D015032:Zinc; D002118:Calcium